Hepatocellular carcinoma (HCC) is one of the most common gastrointestinal (GI) malignancies and a major cause of death worldwide, with limited therapeutic options. Chronic liver diseases are believed to contribute significantly towards the development and progression of HCC due to their role in inflammation and injury. These chronic liver diseases including hepatitis, fatty live diseases (alcoholic and nonalcoholic) are prevalent among our veterans, thus creating a significant financial and medical burden. The only drug currently available to treat HCC is the multikinase inhibitor Sorafenib, which is effective only for a few months. Designing safe and efficient therapeutic approaches that can target additional signaling axes (aberrant in HCC) is critical for treating this deadly form of malignancy. Activation of various oncogenic pathways have been reported in HCC, which include PI3K/AKT/mTOR and Wnt/?-catenin signaling cascades, for which little or no effective therapies are available currently. Of particular importance is the Wnt/?-catenin pathway, since oncogenic mutations of ?- catenin have been reported in a number of adult and childhood liver cancers. Attempts to develop drugs that can effectively target the Wnt/?-catenin axis has been extremely challenging mostly due to the fact that ?- catenin is an intracellular protein without any enzymatic activity, and thus this axis is currently considered undruggable. This is further complicated by the fact that ?-catenin can localize in various cellular compartments and mediate multiple cellular functions some of which are paradoxical. Identification of specific upstream signaling pathways that can modulate ?-catenin axis is thus critically needed to develop targeted therapies for HCC patients with aberrant activation of this axis. Our recent studies revealed a novel signaling pathway mediated by Mixed Lineage Kinase 3 (MLK3), a MAPK Kinase Kinase (MAP3K) that impacts ?- catenin-mediated oncogenic signaling. Our studies indicate that MLK3 can phosphorylate and stabilize ?- catenin in cancer cells, which involves a novel pathway independent of GSK3?. Despite stabilizing ?-catenin, MLK3 inhibited ?-catenin/TCF-dependent transcriptional activity and ultimately resulted in G2/M cell cycle arrest. Based on the preliminary results, we hypothesize that MLK3 activation can successfully antagonize HCC progression via inhibiting conventional Wnt/?-catenin pathway. The current proposal aims to validate our hypothesis utilizing both in vitro (cellular) and in vivo (animal) models while elucidating the fundamental mechanisms by which MLK3 modulates ?-catenin pathway in HCC. The following specific aims are proposed to achieve our goals: (i) Determine the role of MLK3 in modulating Wnt/?-catenin axis in HCC cells, (ii) Elucidate the molecular mechanism of MLK3-induced modulation of Wnt/?-catenin axis in HCC cells and (iii) Determine the role of MLK3 in antagonizing Wnt/?-catenin axis in an in vivo model of HCC. Upon completion of these aims, we expect to establish the in vivo role of MLK3 in antagonizing Wnt/?-catenin axis in HCC, and identify the potential mechanism, which can be utilized for developing targeted therapeutic options in the future. The studies with the phospho-?-catenin mutants are expected to provide novel mechanistic insight revealing how this antagonism is achieved and identify new regulators of Wnt/?-catenin axis. Combined together, this information can be utilized to develop the next generation (more potent) MLK3 agonists to be utilized as monotherapy or in combination therapies to specifically target the ?-catenin axis i HCC patients.